1. Field of the Invention
This invention is directed to papovavirus-derived episomes that replicate efficiently in mammalian cells, yielding stable transfectants having a high episomal copy number and expressing encoded genes at high levels. Papovavirus-derived episomes may be useful in gene therapy strategies to modulate the growth of bladder carcinoma cells.
2. Review of Related Art
One approach to gene therapy of human cancer cells is to introduce vectors expressing antisense sequences to block expression of dominant oncogenes and growth factor receptors. However, high-level expression of the oncogenes requires comparable levels of antisense expression, which presents a considerable technical obstacle, particularly when using expression vectors having a limited potential for achieving multiple copies in stable transfectants. Human cells transduced by retroviral vectors have only one or several copies of integrated retrovirus in stable transfectants. In contrast, hundreds of copies of episomal plasmids can accumulate in stable transfectants because these vectors replicate extrachromosomally. One method to express high levels of antisense transcripts is to utilize episomal plasmid vectors than can replicate extrachromosomally in human cells.
Attempts to produce episomal vectors that will replicate in some types of human cells are reported by the literature. Episomal plasmids have been developed from several DNA viruses, including bovine papilloma virus (BPV) (Sarver, et al., 1981, Mol. Cell. Biol, 1:486-496; DiMaio, et al., 1982, Proc. Natl. Acad. Sci., U.S.A., 97:4030-4034), SV40 (Tsui, et al., 1982, Cell, 30:49914508), Epstein-Barr virus (EBV) (Yates, et al., 1985, Nature, 313:812-815; Margolskee, et al., 1988, Mol. Cell. Biol, 8:2837-2847; Belt, et al., 1989, Gene, 84:407-417; Chittenden, et al., 1989, J. Virol., 63:3016-3025), and BK virus (BKV) (Milanesi, et al., 1984, Mol. Cell. Biol., 4:1551-1560). Each of these episomal plasmids contains a viral origin of DNA replication and a virally encoded early gene that trans-activates the viral origin and allows the episome to replicate in the transfected host cell.
Although EBV-based episomes have been used to efficiently screen cDNA libraries, the EBV system has limited applications to non-lymphoid cell types (Vidal, et al., 1990, Biochim. Biophys. Acta 1048:171-177)), and the EBV replicon is not active in many cell types. Additionally, EBNA-1 is one of several EBV latent genes that immortalize human lymphocytes, and transfection of the EBV-negative BJAB lymphoma cell line by EBNA-1 induces soft agar growth, indicating transformation of the cells. (Konoshita, 1990, Hokkaido Igaku Zasshi, 65:362-375)
Furthermore, stable transfection efficiencies for EBNA-1 negative cell lines transduced by EBV episomal plasmids encoding EBNA-1 (transactivator) and ORI-P (EBV DNA origin) are low, not significantly better than non-episomal plasmids (Yates, et al., 1985; Vidal, et al., 1990. However, if EBNA-1 is expressed in cells prior to transfection, then a subsequent transfection with a plasmid containing ORI-P and a selectable marker can yield stable transfection efficiencies of up to 10% (Margolskee, et al., 1988; Belt, et al., 1989; Yates, et al., 1984, Proc. Natl. Acad. Sci. USA 81:3806-3810; Lutfallia, et al., 1989, Gene 76:27-39). Comparable results have been noted in a related system of COS cell clones expressing high levels of SV-T, which permit efficient replication of SV40 origin-containing plasmids in transient transfectants (Tsui, et al., 1982; Rio, et al., 1985, Science 227:23-28; Chittenden, et al., 1991, J. Virol. 65:5944-5951).
In the COS cell system, however, episomal replication can proceed in a runaway fashion, resulting in up to 104 episomal copies by 48 hours after transfection. Despite efficient episomal replication in transient transfectants, low stable transfection efficiencies have been noted in these studies (Chittenden, et al., 1991; Roberts, et al., 1986, Cell, 46:741-752). Presumably, most transient transfectants die secondary to episome-mediated cell death (Chittenden, et al., 1991; Roberts, et al., 1986).
However, transfection of COS cells by SV40 DNA origin-containing plasmids does produce stable transfectants having episomal plasmids (Tsui, et al., 1982), and it may be possible to control runaway episomal replication by a variety of strategies, including use of replication control regions from other viruses. For example, runaway episomal replication in COS cell clones can be controlled by use of plasmids containing the SV40 DNA origin and regions of the bovine papilloma virus (BPV) replicon (Roberts, et al., 1986; Roberts, et al., 1988, Cell 52:397404). These studies have identified two BPV sequences (NCOR I and NCOR II) that modulate runaway SV40 episomal replication in transient transfectants, and a third trans-suppressing factor encoded by 5xe2x80x2 sequences in the El open reading frame. Hybrid plasmids encoding the SV40 DNA origin and a 2113 bp EcoRI fragment of BPV have substantially higher stable transfection efficiencies than pSV-NEO (Roberts, et al., 1986). A DNA homology search failed to identify similar NCOR sequences in the BKV or SV40 replicon.
Thus, there remains a need for vectors which will replicate episomally in a controlled fashion in mammalian cells for gene therapy applications. In particular, there is a need for vectors that will replicate episomally in human cells without transforming the cells.
It is an object of this invention to provide a vector which will reproduce episomally in a mammalian cell without transforming the cell.
It is another object of this invention to provide a method for gene therapy whereby a foreign gene, encoded on a vector that replicates episomally in high copy number without transforming transfected cells, is expressed in a mammalian cell transfected by the episomal vector.
It is still another object of this invention to provide a mutant form of papovavirus large T antigen that is replication-competent and transformation-negative.
In order to achieve these and other objects, the present invention provides a mammalian vector which is replication-competent and transformation-negative, the vector comprising at least one papovavirus origin of replication, preferably the origin from SV40 or BK virus, and a DNA sequence encoding a mutant form of papovavirus large T antigen which contains a replication-competent binding site for the origin of replication but which is negative for binding to at least one of wild-type p53 or retinoblastoma tumor suppressor (RB) gene products, preferably both, the DNA sequence being operatively linked to a homologous or heterologous promoter. In alternative embodiments of the vector, the DNA sequence encoding a mutant form of papovavirus large T antigen is operationally linked either to a papovavirus early promoter, to a promoter which is inducible, or to a promoter which is under hormonal control.
In another embodiment, this invention provides a method of expressing a foreign gene in a mammalian cell comprising transfecting the mammalian cell with a replication-competent, transformation-negative vector comprising at least one papovavirus origin of replication, a first DNA sequence encoding a mutant form of papovavirus large T antigen which contains a replication-competent binding site for the origin of replication but which is negative for binding to at least one of wild-type p53 or retinoblastoma tumor suppressor gene products, the DNA sequence being operatively linked to a first promoter, and a second DNA sequence encoding the foreign gene operatively linked to a second promoter; and expressing the foreign gene in the transfected cell. In preferred embodiments of this method, the papovavirus origin of replication is either the BK virus origin of replication or the SV40 origin of replication, and the mutant form of papovavirus large T antigen is a mutant SV40 large T antigen that binds to both SV40 and BK virus origins of replication but is negative for binding wild-type p53 and also negative for binding to retinoblastoma tumor suppressor gene product. In alternative embodiments, the mammalian cell is transfected by the vector in vitro, then the cell is introduced into a mammal and the foreign gene is expressed in vivo, or the vector is administered to a mammal and cells of the mammal are transfected in vivo, the foreign gene being expressed by these cells.
In another embodiment, the invention provides a DNA sequence encoding a mutant form of SV40 large T antigen which contains a replication-competent binding site for SV40 origin of replication but is negative for binding to wild-type p53 and is also negative for binding to retinoblastoma tumor suppressor gene product. In a preferred embodiment, residue 107 of the mutant form of SV40 large T antigen encoded by the DNA sequence is lysine and residue 402 is glutamic acid.
A highly efficient episomal expression vector that replicates extrachromosomally in human cells has been developed. We have demonstrated that replication-competent, transformation-negative SV40 large T antigen mutants can successfully drive replication of plasmids containing the SV40 DNA origin or BK virus origin of DNA replication. A preferred vector is derived from BK virus (BKV), a small DNA virus having significant homology to SV40. The properties of BKV episomes characterized in stable bladder carcinoma cell line transfectants have shown that these vectors replicate extrachromosomally for at least 5 months; achieve a high stable copy number (150) without inducing episome-mediated cell death; have a very low rate of integration; transcribe genes in proportion to their copy number; are efficiently transferred to daughter cells during cell division; can be shuttled from Hirt supernatant DNA to bacteria; and even persist in bladder cell transfectants for several months without selection pressure. These properties demonstrate the feasibility of using this vector system to transfer genes to human cells.
This invention makes a significant advance in episomal vector technology by developing replication-competent, transformation-negative mutants of papovavirus large T antigen to drive replication of plasmids containing papovavirus origins of DNA replication, such as the SV40 or BKV DNA origins. Since replication-competent, transformation-negative mutants for other DNA origin transactivators (such as EBNA-1) are not currently available, this episomal expression system has the unique feature of permitting efficient episomal replication without induction of transforming properties in the host cell. This advance enables development of safe and efficient episomal vectors for human gene therapy applications. This episomal vector system may also have widespread in vitro applications, such as development of a cancer tumor progression assay, and has the potential to significantly advance development of efficient episomal vector systems for cDNA library cloning and in vitro expression of heterologous genes in human cells.